In this project, we are developing new diagnostic and therapeutic approaches to immune dysregulatory diseases. First, we have pursued novel therapeutic approaches focusing on multiple sclerosis (MS) as well as new immunological diseases for which we have determined the genetic basis. Our principle approach is to use antigen itself to program the specific cognate T cells to die through apoptosis via a process termed restimulation-induced cell death (RICD). The death is clonally specific and represents a way to eliminate disease-causing T cells by using antigen. Additionally, depending on the molecular and genetic mechanism of disease, we also investigate using other small molecules or protein-based therapeutics. The key is to precisely tailor the therapy to the pathophysiological mechanism of the disease so that it will be highly effective. This is the overarching goal of precision medicine. At the present time, we have chosen to focus on developing new, highly effective therapeutics against MS using immunological and genetic approaches. Evidence suggests that myelin proteins antigens are targets of the autoimmune attack, but how the specificities determine disease outcome in progressive and relapsing-remitting MS is unclear. By programming the T cells that recognize such antigens to die, the effect of eliminating these cells on the disease can be demonstrated. We are also studying new highly sensitive diagnostic tests to detect reactive T cells against these antigens to determine if these can provide an early warning system of autoimmune attack. Although twin studies show that there is a significant genetic component to MS susceptibility, there is little concrete knowledge about the genes or pathways involved. Genome-wide association studies (GWAS) have unearthed a variety of single nucleotide polymorphisms (SNPs) that are statistically associated with disease. The International MS Genetics Consortium has performed numerous GWAS but the number of identified SNPs with strong association with MS has been much lower than anticipated. However, these studies that the correlated SNPs are in or around mainly immune genes rather than central nervous system genes. Nonetheless, the implication of specific pathways in various subtypes of MS has not been achieved. Therefore, we have concluded that approaching the problem by searching for Mendelian or de novo genetic variants may provide more definitive information about pathogenesis. Using next generation sequencing (NGS) techniques such as whole exome sequencing (WES) and whole genomic sequencing (WGS) we are now capable of identifying causal genes in rare and severe disorders that do not have enough fitness to be inherited broadly in the population and detected by GWAS. Moreover, these highly penetrant and deleterious variants likely contribute strongly to de novo and Mendelian forms of autoimmune disease as well as susceptibility to common autoimmune diseases. Identification of rare genetic variants generally provides key information about disease mechanisms, biological pathways, and novel avenues for clinical treatments. We have established a collaboration with investigators at the University of British Columbia, Canada who have assembled over 450 Multiplex MS families: 1.Families with 3 or more affected members with MS (400 families); 2. Families with index cases with age of onset < 12 years old (extremely rare and available under this study); 3. Families with MS combined with another autoimmune disease (type I diabetes, systemic lupus erythematosus, rheumatoid arthritis, inflammatory bowel disease). These patients, family members, and relatives will be subjected to WES and possibly WGS through a collaboration with the Regeneron Sequencing Center. We expect that gene variants found by NGS in these patients, particular in the first two categories, will unveil major pathogenic pathways for MS. In order to better understand the molecular underpinnings of the RICD regulatory mechanism, we have carried out extensive molecular investigations of how TCR stimulation directs cells to a death pathway instead of simply activation. We have found that cycling T cell blasts become susceptible to restimulation-induced cell death (RICD) by redirecting the NF-kB-activating pathway. We carried out a chemical genetics screen that revealed a critical role for PI-3K activating a novel pathway to the induction of I-kappaB kinase (IKK). This transduces a death signal independently of Fas or other tumor necrosis factor superfamily receptors. We have complemented this by carrying out a CRISPR screening using RICD as a selection. Interestingly, NF-kB itself and new transcription/translation were found to be dispensable for RICD in activated but not nave T cells. This is apparently due to the fact that the caspase-3 dimer is partially cleaved but not fully processed in activated cycling T cells blasts suggesting the molecular pathway has been engaged. TCR restimulation then introduces the full processing of caspase-3 causing cellular demise. Further experiments are being directed at determining the precise substrate of IKK. This may shed light on a key molecular process that will be useful for antigen-induced treatment of autoimmune diseases and, potentially, immunotherapy for cancer. We have also moved our research in the direction of therapeutics particularly for a unique disorder we have termed p110delta activating mutations causing Senescent T cells, Lymphadenopathy, and Immunodeficiency (PASLI) disease. We have studied the germline, heterozygous, dominant, gain-of-function mutations in the p110delta catalytic subunit of PI3K in nine patients from seven unrelated families and also families with severe mutations in the regulatory subunit of PI3K. We have investigated specific p110delta enzyme inhibitors in PASLI patients and entered into a Cooperative Research and Development Agreement with Novartis to carry out a clinical trial testing their inhibitor of p110delta, a compound called leniolisib (CDZ173), in the NIH clinical Center. Preliminary evidence shows that leniolisib is a potent and selective inhibitor of p110delta and can inhibit the wild-type as well as overactive forms of the enzyme thereby potently suppressing disease with few or no side effects. A larger double-blind, placebo -controlled study is underway to move towards approval as a new drug with FDA. Lastly, we expanded our project to include therapeutics for IL-2 receptor (IL-2R) complex, which plays a central role in control of the immune response by integrating signals from the key cytokines. We are the first to report monogenic cause of human IL-2R beta chain deficiency. We studied autosomal recessive IL-2R beta chain deficiency in four families. Clinical hallmarks of the disease include prominent immune dysregulatory phenomena and susceptibility to respiratory and herpesvirus infections. Three unique mutant alleles were found in the four families, with each allele causing IL-2R beta chain deficiency by different biochemical mechanisms. These discoveries provide insight into one of the principal signaling pathways of the immune activation and peripheral tolerance and should prompt prenatal screening of IL-2R beta chain mutations and genetic counseling in families at risk.